In situ hybridization for fungal ribosomal RNA sequences using locked nucleic acid (LNA) probes

Abstract

LNA are modified RNA nucleotides where a ribonucleoside is linked between the 2′‐oxygen and the 4′‐carbon atoms with a methylene unit. LNA nucleotides exhibit increased thermal stability. ISH utilizing LNA probes targeting rRNA sequences has not been described. ISH using 3′ biotin‐labeled LNA probes (50% DNA/50% LNA) targeting Aspergillus, Zygomyces, Candida, and Blastomyces rRNA sequences was performed on culture proven paraffin‐embedded cases of Aspergillus, Zygomyces, Candida, and Blastomyces using the Microprobe staining system. The tissues were dewaxed, cleared, rehydrated and digested with pepsin. The probes were applied to the tissue sections, heated briefly, hybridized at 50°C and washed with 2XSSC. The hybrids were detected using anti‐biotin‐alkaline phosphatase followed by Permanent Red chromogen. The ISH assays detected the appropriate fungal pathogens in FFPE tissues. ISH using LNA probes targeting fungal rRNA sequences can be used for speciating fungal pathogens in paraffin‐embedded tissues. These tests could be useful when fungal pathogens are observed in tissue but cultures are negative or have not been performed.